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The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b 558 (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91 phox and p22 phox to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91 phox precursor, gp65, was processed to gp91phox within 4 -8 h of chase, unassembled gp65 and p22 phox monomers were degraded by the cytosolic proteasome. gp65 associated with p22 phox post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22 phox coprecipitated with unglycosylated gp91 phox primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22 phox was unaffected. In succinyl acetone-treated cells, p22 phox and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22 phox heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22 phox , a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp9l-phox encompassing residues S8TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E-5mSGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP'60 and G177GPPGGP'83). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp9l-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ40were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 ,LM and 230 ,uM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91-and p22-phox and other regions of gp9l-phox nearer to the N terminus.

The NADPH oxidase cytochrome b 558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91phox , and a nonglycosylated 22-kDa subunit, p22phox . The role of heme in cytochrome b 558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22 phox and the mature 91-kDa form of gp91 phox but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b 558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91 phox in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22 phox mRNA, demonstrated that p22 phox was not required for maturation of gp91 phox carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22 phox increased the abundance of the mature gp91 phox glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b 558 assembly and provide further support for the concept that stability of p22 phox and the mature gp91 phox subunit is increased by heterodimer formation.

Flavocytochrome bs« is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp9lP hOX and p22P hox subunits. Human flavocytochrome b sss is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22P hox • 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp9lP h0 X. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp9lP h0 X. In addition, 7D5 immunoprecipitated the -58 kDa unglycosylated gp91 phOX protein from solubilized membrane fractions of tunicamycintreated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91PhOX but not p22 phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91 phOX and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome bsss-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome bsss.

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Seldom could metals and alloys maintain excellent properties in cryogenic condition, such as the ductility, owing to the restrained dislocation motion. However, a face-centered-cubic (FCC) CoCrFeNi highentropy alloy (HEA) with great ductility is investigated under the cryogenic environment. The tensile strength of this alloy can reach a maximum at 1,251±10 MPa, and the strain to failure can stay at as large as 62% at the liquid helium temperature. We ascribe the high strength and ductility to the low stacking fault energy at extremely low temperatures, which facilitates the activation of deformation twinning. Moreover, the FCC→HCP (hexagonal close-packed) transition and serration lead to the sudden decline of ductility below 77 K. The dynamical modeling and analysis of serrations at 4.2 and 20 K verify the unstable state due to the FCC→HCP transition. The deformation twinning together with phase transformation at liquid helium temperature produces an adequate strain-hardening rate that sustains the stable plastic flow at high stresses, resulting in the serration feature.

The redox center of the phagocyte NADPH oxidase is flavocytochrome b 558 , a transmembrane protein with two subunits, gp91 phox and p22 phox . In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91 phox precursor (p65). Expressing the gp91 phox cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91 phox transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22 phox was found in subcellular compartments containing the mature 91-kDa form of gp91 phox and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91 phox and p22 phox but had little or no impact on p65. These studies demonstrate (a) gp91phox is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.